The last two decades have seen vast progress in the fields of immunochemistry and molecular biology, and this is clearly reflected in the volume of relevant scientific and patent literature appearing during this period. Among areas for which the present invention is of relevance, an area of particular growth has, for example, been that of qualitative and/or quantitative assays involving the use of immunoreactive species, i.e. antigens, haptens or antibodies.
One such area is that of immunohistochemical/cytochemical detection procedures, the purpose of which is normally the localization of antigenic determinants present in tissues or in/on cells via immunochemical reaction of these antigenic determinants with specific so-called primary antibodies which react only with the target antigenic determinants. The primary antibodies are either labelled with appropriate labels (e.g. enzymes, fluorescent groups or heavy atoms), or they are themselves further detected via an immunochemical reaction with specific so-called secondary antibodies which react with the primary antibodies; in the latter case it is the secondary antibodies which are labelled with appropriate labels (e.g. enzymes, fluorescent groups or heavy atoms). Alternatively, the immunochemical reaction between target antigenic determinants and primary antibodies is detected via an immunochemical reaction with a specific so-called link antibody which has the property of reacting simultaneously with (i) the primary antibodies and (ii) an antibody to which enzymes have been attached via an immunochemical reaction or via covalent coupling.
As a further alternative, the immunochemical reaction between target antigenic determinants and primary antibodies, or between primary antibodies and secondary antibodies, is detected by exploiting the binding which occurs between certain pairs of complementary molecules other than antigens and antibodies; an example of such a complementary pair is biotin and streptavidin. In this approach, one member of the complementary pair is attached to the primary or secondary antibodies and the other molecule is combined with suitable labels (e.g. enzymes, fluorescent groups or heavy atoms).
In procedures of this type, a specimen in the form of a sample of thinly cut tissue or a sample of cells (typically from a body fluid) is affixed to a glass slide. The applied specimen is then normally treated with various chemicals to facilitate the subsequent immunochemical reactions. The specimen is then subjected to treatment with a labelled or non-labelled primary antibody, as appropriate, whereupon the antibody becomes immunochemically bound to the antigen in question in/on the specimen. After removal of excess antibody by suitable washing of the specimen, the antibody bound to the antigenic determinant is detected by treatment with appropriate reagents, depending on the choice of the visualization system (as described previously, above) in conjunction with suitable washing procedures. After removal (by washing) of the excess labelled reagent from the chosen visualization system, the specimen is subjected to a treatment as follows, depending on the label in question:
(i) in the case of enzyme labels, the specimen is treated with a substrate (colour developing reagent). The enzyme reacts with the substrate, leading to the formation of a coloured insoluble deposit at and around the location of the enzyme;
(ii) in the case of a heavy metal label such as gold, the specimen can be treated with a so-called enhancement reagent containing silver. Silver metal is then precipitated as a black deposit at and around the location of the gold.
(iii) in the case of fluorescent labels a developing reagent is normally not needed.
After a washing step, some of the constituents of the specimen can then be coloured by a chemical dye which gives a suitable contrast to the colour given by the label in question. After a final washing step, the specimen is coated with a transparent reagent to ensure a permanent record for the examination.
The visualization of the labels (indirectly expressing the localization and amount of target antigenic determinants) is performed as follows:
(i) light microscopic examination (in the case of enzyme labels);
(ii) light or electron microscopic examination (in the case of heavy metal labels);
(iii) fluorescence microscopic examination, using irradiated light of a suitable wavelength (in the case of fluorescent labels).
The period in question has also seen, for example, the emergence and development of assays of the so-called ELISA (Enzyme-Linked Immuno-Sorbent Assay) type, in which an antigen, hapten or antibody is detected by means of an enzyme which is covalently coupled (also denoted linked or conjugated) either (when an antigen or hapten is to be determined) to an antibody which is specific for the antigen or hapten in question, or (when an antibody is to be determined) to an antibody which is specific for the antibody in question. In "traditional" ELISA, the antigen, hapten (the latter generally in the form of a conjugate with, e.g., a protein) or antibody to be detected/determined is normally bound or immobilized by allowing it to bind immunochemically to (i) a so-called "catching" antibody in the case of antigen or hapten determination or (ii) an antigen in the case of antibody determination, each of which is attached (generally by non-covalent adsorption) to the surface of an appropriate material, such as polystyrene in the form of beads or microtiter trays, and the appropriate enzyme-linked specific antibody is then allowed to bind to the immobilized species which is to be detected/determined; the amount of bound specific antibody, and thus the amount of immobilized species, is then determined by adding a substance which is a substrate for the linked enzyme and which, upon enzymatic decomposition, results in the development of a characteristic colour, the intensity of which (measured, for example, by spectrophotometry or simple colorimetry/comparimetry) is thus related (normally proportional) to the quantity of the species of interest which is to be determined. Examples of preferred enzymes for use in assays of this type (as well as in immunohistochemical procedures) are peroxidases, e.g. horseradish peroxidase, alkaline phosphatase, glucose oxidases, galactosidases and ureases.
Immunochemical assays of a type analogous to ELISA but employing other means of detection, e.g. the use of specific antibodies to which fluorescent or luminescent marker molecules are covalently linked, have also undergone considerable development in the same period, and the emergence of so-called "time-resolved fluorescence" is a good example: In this technique the marker or label is generally either Eu.sup.3+ or a europium chelator (although certain other lanthanide species or lanthanide chelators have also been employed), and a fluorescent europium chelate can then be formed by adding an organic chelator or Eu.sup.3+, respectively. In contrast to most of the more traditional fluorescent marker species, e.g. fluorescein, which generally have fluorescence lifetimes of about 100 nanoseconds (nsec) or less, the fluorescence lifetime of lanthanide chelates is generally in the range of 100-1000 microseconds (.mu.sec); by making use of a pulsed light source and a time-gated fluorometer, the fluorescence of these compounds can be measured in a time-window of about 200-600 .mu.sec after each excitation. A main advantage of this technique is the reduction of background signals which may arise from short-lived fluorescence of other substances present, for example, in the analysis sample, in or on the material of microtiter wells, in cuvettes or the like, or elsewhere in the measurement system.
A further group of procedures which employ immunochemical detection techniques, and which should be mentioned in the context of the invention, are "immunoblotting" procedures, examples of which are the so-called "dot blot" and "western blot" procedures: In the western blot procedure, which is employed for the analysis and identification of antigenic polypeptides or proteins, the proteins/polypeptides in a mixture thereof are separated by polyacrylamide gel electrophoresis and then transferred electrophoretically ("blotted") to a sheet of nitrocellulose or chemically treated paper to which the proteins/polypeptides bind in a pattern identical to that in the gel. The appropriate specific antibody is then added, followed by a labelled second antibody against the first antibody or labelled protein-A (labelled with, for example, a radioisotope, fluorescent dye, enzyme or colloidal gold). The location of the label (and thus the presence of the particular antigen) is then detected in the appropriate manner outlined previously.
As far as developments in the general field of molecular biology are concerned, one non-immunochemical area of significance has been that of hybridization techniques in connection with gene structure analysis: In "traditional" hybridization techniques, a particular nucleotide sequence (also known as a "probe" or "gene probe") is labelled with an appropriate marker or label, e.g. a radioactive isotope, and is then added to a sample of a nucleic acid of interest, e.g. a sample in the form of part of intact cells or in the form of isolated DNA or RNA fragments. The sample can either be free in solution or immobilized on a solid-phase substrate. If the probe and the nucleic acid sample hybridize by formation of a strong, non-covalent bond between them, it can reasonably be assumed that a nucleotide sequence essentially identical to that of the probe is present in the nucleic acid. The marker or label on the probe thus provides a means of establishing whether hybridization has occurred, and for determining the amount of DNA/RNA sample present.
The so-called "Southern blot" method for the detection of rare DNA fragments in a complex mixture of DNA is an example of a procedure employing the technique of hybridization: Gel electrophoresis is used to separate the various fragments, which are then denatured and transferred by blotting to nitrocellulose sheet. The fragments are then hybridized to an appropriate radioactively labelled probe, and their position is revealed by autoradiography. Analogous procedures have been devised for RNA and, as already mentioned ("western blot", vide supra), for protein or peptide antigens.
Hybridization techniques have been of great importance in biochemical genetics for an understanding of the relationship between nucleotide sequences and their function, and they provide an important diagnostic tool for the detection, for example, of genetic defects, or of infectious agents such as viruses or bacteria.
Owing primarily to the health hazards posed by the use of radioisotopes in hybridization procedures of the above types, attempts have been made to replace them with more innocuous (and, generally, more readily available) markers or labels. However, attempts made until now, for example using biotinylated probes to be detected by means of, e.g., enzyme-labelled reagents, have resulted in attendant loss of sensitivity relative to that obtainable using radioactively labelled probes.
The above-mentioned problem of poor detection sensitivity when using, in particular, non-radioactive labels, applies not only to hybridization techniques, but also to immunochemical detection or assay procedures or when immunochemical detection is used to amplify a hybridization reaction. In other words, the lower detection limit may prove to be inadequate for the unambiguous detection or accurate quantitative determination of low levels of, e.g., antigens or antibodies or nucleic acids. This may, for example, be due to the intensity of colour or fluorescence in an immunochemical or hybridization procedure of one of the above-mentioned types being too low, chiefly as a consequence of the fact that normally only one or, at best, a very few (generally less than five) molecules of enzyme or marker species can be linked, e.g., to each specific antibody molecule or to each molecule of the nucleotide sequence of the probe. Furthermore, for each marker species which is attached (conjugated) to, e.g., an antibody molecule, and particularly if the marker species impart a net positive or negative charge to the antibody/marker conjugate, there is an increasing risk of a deleterious influence on the natural ability of, for example, the antibody to participate in the correct immunochemical binding reaction of interest; furthermore, the presence of a net positive or negative charge on such a conjugate increases the risk of undesirable non-specific binding of the conjugate to other materials or species in the system.
In the immunochemical field in particular, considerable effort has been devoted to devising ways to enhance the sensitivity of immunochemical assay procedures, and one approach which has achieved a good measure of success involves the attachment of an immunochemically reactive species, e.g. an antibody, and a plurality of enzyme molecules, fluorescent marker molecules or the like to one and the same backbone or carrier, e.g. polymeric carrier. There are numerous patent documents relating to this kind of approach, and making use of either soluble or insoluble carriers. Generally speaking, in applications of the type outlined above, the use of soluble carriers is to be preferred, since the presence of the carrier (with the coupled immunochemically reactive species and enzymes/marker molecules or the like) in homogeneous solution rather than as a heterogeneous phase, together with the relatively great conformational flexibility of such species in the solution phase, vastly enhances the rate and, in general, the extent of immunochemical reactivity with the immunochemical counterpart species which is to be detected or determined; in immunohistochemical applications the use of soluble carriers is virtually essential, since good tissue contact or penetration is necessary in order to ensure optimal access to the immunochemically reactive moieties or epitopes located on or within the tissue. Moreover, it is generally much easier to remove (e.g. by washing or flushing) carrier-borne immunochemically reactive species, e.g. carrier-borne antibodies, which have not (e.g. owing to "saturation" of available binding sites) become bound to immunochemical counterparts, e.g. to antigens attached, for example, to the surface of an microtiter tray, immunoplate or the like, when the carrier-borne species in question are soluble than when they are insoluble or colloidal.
The present invention represents a significant advance with respect, inter alia, to the enhancement of flexibility, sensitivity and reliability of all of the various types of detection and assay procedures outlined by way of example above. The invention can also be exploited, for example, to reduce--without loss of sensitivity--the number of successive "layers" of immunoreactive components (antigen, antibody, anti-antibody etc) which would otherwise be required in the performance of, e.g., a traditional ELISA or histochemical procedure. Other advantages associated with the invention will become apparent from the present specification and the working examples given herein.
Since the present invention relates, as already indicated, to water-soluble reagents and conjugates, and to their preparation and use, the following outline of a number of pertinent patent documents is confined to disclosures in which soluble carriers are employed:
European patent 0 077 671 relates inter alia to a water-soluble, non-cross-linked and non-primary-amine-containing polymer to which is conjugated a marker substance. The polymer/marker substance conjugate has a negative or zero charge, and to the polymer part of each molecule thereof there is attached "only one immunological homologue" (antigen or antibody). The original European patent application (EP 0 077 671 A1) does not restrict itself to "only one" immunological homologue molecule, but neither is there any specific mention of more than one immunological homologue. The preferred water-soluble polymers in the latter patent/patent application are polyacrylic acid, polymethacrylic acid, polyacrylamide, polyvinyl alcohol, polyallyl alcohol, polymer combinations of the foregoing, hydroxyethyl-cellulose, hydroxypropyl-cellulose, natural water-soluble polymers and synthetic water-soluble polymers. The molecules of marker substance may be incorporated ab initio in the polymer by co-polymerizing a relatively small percentage of a suitable monomer incorporating the marker substance (e.g., in the case of a fluorescent marker substance, a monomer produced by the reaction of fluoresceinamine with acryloyl chloride in equimolar amounts) with the monomers which form the basis of the polymer backbone (e.g. acrylic acid and acrylamide). Other mentioned methods for attachment of marker substances to the polymer include making use of an activated group on the marker substance, and employing an "external activating agent". As regards the attachment of the immunological homologue to the polymer backbone, the following examples are given:
(i) an acrylic acid/acrylamide copolymer incorporating ca. 1% of a copolymerized monomer produced by reaction of fluoresceinamine with acryloyl chloride is activated by reaction with carbonyl diimidazole and N-hydroxysuccinimide, and the resulting activated polymer/marker substance conjugate is then reacted with a monoclonal antibody;
(ii) a monoclonal antibody which has been biotinylated by reaction with biotinyl-N-hydroxysuccinimide is added to a conjugate formed by reaction of avidin with the activated polymer/marker substance conjugate prepared as outlined in (i), above; attachment of the monoclonal antibody to the polymer backbone in this case is via the strong, but non-covalent, binding interaction between the biotin groups covalently bound to the antibody, and avidin moieties covalently conjugated to the polymer/marker substance.
There appears to be no disclosure in this patent relating to the use of dextrans as the polymer carrier, or of divinyl sulfone, or a moiety derived therefrom, as an activating reagent or coupling/bridging moiety via which a marker substance or an immunological homologue may be attached to the polymer.
U.S. Pat. No. 4,152,411 relates to a labelled "spine tool" for determination of a component of the antigen/antibody reaction, preferably making use of polymers with amide bonds between the individual units of the polymer, for example polylysine, other homopoly(amino acids) or polypeptides; on average, only one molecule (hapten, antigen or antibody) to be "labelled" is attached to the polymer "spine tool". The specification mentions tolylene-2,4-diisocyanate, glutaraldehyde and carbodiimide as candidates for activation reagents by which the molecule to be "labelled" may be coupled to the polymer carrier, and two specific examples illustrate the use of 1-ethyl-3-(3-dimethyl-amino-propyl)-carbodiimide for the coupling of a hapten, viz. thyroxine. Whilst a number of possible labelling or marker species are mentioned, including numerous fluorescent substances (such as fluorescein) and enzymes (such as peroxidases), details of the manner in which these are to be attached to the polymer carrier are given only in the case of one specific enzyme, namely horseradish peroxidase (HRP), which in the example in question is coupled to polylysine/thyroxine conjugate via the initial oxidation of the diol moiety of HRP using sodium periodate; the resulting diketone moiety of the oxidized HRP is then allowed to react with the latter conjugate (via an amino function of the polylysine part) to form a Schiff base intermediate, which in turn is then reduced, e.g. using sodium borohydride, to give the HRP-labelled polylysine/thyroxine conjugate. This particular type of labelled conjugate ("diagnostic marker spine tool") is stated as being ". . . amazingly stable upon storage for extended periods. It is considered feasible to store such a tool for up to three or six months or more in inert atmosphere, e.g. under nitrogen, and in the absence of moisture."
The possibility of using polysaccharides, including dextran, as the basis for the "spine tool" is briefly mentioned in the specification, but there is no indication of the detailed manner in which attachment of the molecules of interest to dextran should take place. Neither is there any indication of any advantages associated with the use of dextran for this purpose.
EP 0 010 405 A1 relates, inter alia, to an immunochemical assay reagent comprising a carboxyl-containing, water-soluble, mono-olefinic polymeric compound combined (i.e. coupled) with a hapten or a chemically modified product thereof, and to a method for the immunochemical determination of a hapten using such a reagent. There appears to be no mention of the use of polymers related to dextran in the context of this patent application.
Chemical strategies which are mentioned in connection with the coupling of a hapten to the polymeric compound are: the use of carbodiimides (e.g. dicyclohexylcarbodiimide), carbonyldiimidazole or diphenylphosphoryl azide (DPPA); and the intermediate formation of mixed acid anhydrides (formed, e.g., using a chloroformic acid ester such as isobutyl chloroformate), active esters (formed, e.g., using N-hydroxysuccinimide), azides (formed using hydrazine followed by nitrous acid) or acid chlorides (formed, e.g., using thionyl chloride or phosphorus oxychloride).
The immunochemical assay reagents in question show "very high stability in the form of an aqueous solution . . . and can fully withstand storage at room temperature".
There appears to be no mention of the possibility of coupling an immunologically active species (antigen, antibody) other than a hapten to the polymeric material in question.
U.S. Pat. Nos. 4,166,105 and 4,169,137 (to Hirschfeld and to Hirschfeld et al., respectively) relate to antigen-detecting reagents and dye-tagged reagents, respectively, comprising a primary amine-containing polyfunctional polymer backbone, e.g. a polyethyleneimine backbone, and attached marker molecules (such as a plurality of fluorescent dye molecules); the reagents according to the former patent further comprise an antibody specific for the antigen to be detected, whilst the reagents according to the latter patent further comprise a "first reactant", particularly an antibody. Working examples given in both patents demonstrate an average of at most one antibody molecule attached to the polymer backbone. The use of a dialdehyde, in particular glutaraldehyde, as coupling reagent for coupling marker molecules and antibody/first reactant to the polymer backbone is preferred, there being no mention of the possibility of using divinyl sulfone-based coupling.
EP 0 135 071 A2 relates, inter alia, to chemiluminescent-labelled hapten conjugates comprising a chemiluminescent group (e.g. a group derived from luminol or a derivative thereof), a chain polymeric "attachment group" (in German: Verknupfungsgruppe) and a hapten; the attachment group has repeating functional groups, and for each mole of attachment group there are several moles of luminescent group and several moles of hapten, preferably at least 10 moles of each per mole of attachment group.
Examples of chain polymers which are briefly mentioned in the specification include peptides, proteins, glycoproteins, glycolipids and carbohydrates, including polysaccharides such as dextrans, and divinyl sulfone is briefly mentioned among examples of coupling reagents for coupling of chemiluminescent groups or haptens to repeating functional, reactive groups (Such as amino, carboxy, carbonyl, "thionyl" or hydroxy groups) on the polymer. However, the only working examples provided relate to the preparation and use, in a chemiluminescence assay, of luminol/hapten/protein conjugates based on the proteins (polymers) transferrin and porcine thyreoglobulin, and employing a carbodiimide and succinic anhydride, respectively, as coupling reagents for coupling of the haptens to the proteins.
There is no further disclosure nor any working example in this document relating to the use of, or any advantages associated with the use of, the coupling chemistry preferred in the context of the present invention, viz. coupling based on moieties derived from divinyl sulfone; neither does there appear to be any clear indication of the stability or shelf-life of the disclosed conjugates, either in the solid state or in solution.
In relation to the conjugates disclosed in the documents reviewed above, the reagents/conjugates according to the present invention distinguish themselves, for example, as described in the following:
Firstly, as is well documented by the working examples given herein (vide infra), water-soluble reagents and conjugates of the present invention have been found to possess unexpectedly and extraordinarily high stability/shelf life in aqueous solution at moderate (non-extreme) pH values, not only at low temperatures but also at temperatures somewhat above normal ambient temperatures. It is particularly noteworthy that this is true of:
(i) water-soluble reagents of the invention which have no molecular species [as defined herein; (vide infra)] coupled thereto, i.e. reagents of the invention which comprise a water-soluble polymeric carrier molecule to which there are covalently attached one or more groups or moieties derived from divinyl sulfone, one end of each of which is attached to the carrier molecule via a covalent linkage formed between one of the two vinyl groups of divinyl sulfone and a reactive functionality present on the carrier molecule, and the other end of which retains a free, "dangling" vinyl group which is capable of subsequent reaction with an appropriate molecular species having a suitably reactive functional group; and
(ii) water-soluble conjugates of the invention which comprise a water-soluble polymeric carrier molecule to which are covalently attached, via linking groups derived from divinyl sulfone, one or more molecular species, the polymeric carrier molecule further having covalently attached thereto one or more divinyl-sulfone-derived moieties having a free, reactive vinyl group.
As documented in the working examples herein, the present inventors have found that the inherent reactivity of the free vinyl groups present in the latter types of water-soluble reagents and conjugates of the invention is suppressed at pH values close to neutrality, whereas these groups exhibit very high reactivity at alkaline pH values, e.g. at a pH in the region of about 9-11.
Furthermore, preliminary results obtained by the present inventors also indicate that water-soluble conjugates of the invention which are substantially "saturated" with respect to the possibility of covalent attachment of further molecular species (i.e. conjugates according to the invention which have molecular species covalently attached to the polymeric carrier molecule via linking groups derived from divinyl sulfone, but which substantially lack divinyl-sulfone-derived moieties having free, reactive vinyl groups) also possess similarly high stability/shelf life in aqueous solution at moderate pH values.
The above-described long-term stability exhibited by reagents and conjugates of the present invention thus makes it possible, as already indicated (vide supra), to market such reagents or conjugates in pre-prepared form, e.g. in the form of a kit which might include (where relevant) instructions and, possibly, supplementary chemical reagents for carrying out the appropriate further chemical procedures; the purchaser can thus (i) subsequently attach desired molecular species to pre-prepared, chemically reactive reagents or conjugates of the invention to prepare, for example, assay reagents or conjugates which are tailored to meet specific requirements, or (ii), in the case of pre-prepared, "saturated" conjugates (vide supra) according to the invention, use a pre-prepared conjugate according to the invention directly in a relevant detection or assay procedure.
Secondly, as is apparent from the working examples provided herein (vide infra), in relation to the size of the polymeric carrier molecule the reagents and conjugates according to the present invention, notably the most preferred reagents and conjugates of the invention in which the polymeric carrier molecule is a dextran, are capable of a high degree of loading with molecular species while at the same time remaining water-soluble. Furthermore, taking into account the relatively moderate content of reactive groups (i.e. reactive groups derived from divinyl sulfone, and via which covalent attachment of molecular species takes place) on the polymeric carrier molecules of the intermediate reagents employed in the present working examples in question, and given the fact that appreciably higher contents of reactive groups are, as is well documented in other working examples herein, attainable, it is envisaged that considerably higher levels of loading with molecular species are achievable, for example such that covalent attachment of several thousands of molecular species of low molecular weight, or up to of the order of a thousand molecular species of relatively high molecular weight, per carrier molecule is achievable, depending of course on the steric bulk and/or molecular weight of the molecular species in question, and on the size or molecular weight of the polymeric carrier molecule.
Thirdly, not only is loading of the carrier molecule with a plurality of, for example, hapten species (as in EP 0 135 071 A2) or fluorescein groups (as in U.S. Pat. No. 4,166,105 and U.S. Pat. No. 4,169,137) possible according to the present invention, but it is equally well possible to attach a plurality of molecules of antigens, antibodies, enzymes, gene probes, avidin or other types of molecular substances which will be apparent from the disclosure herein. Particularly noteworthy is the ability according to the invention to attach a plurality of antibodies to a water-soluble polymeric carrier molecule as employed within the context of the invention; on the basis of the patent and scientific literature known to the present inventors and relating to water-soluble reagents or conjugates of a type relevant in relation to the present invention, it would appear that there has either been technical prejudice with regard to the feasibility or desirability of achieving attachment of more than one antibody molecule (or, at best, more than a very few antibody molecules) to the carrier molecule, or, perhaps, that attempts to achieve this have generally failed. The use of an antibody-bearing conjugate according to the invention (bearing a plurality of antibodies and a plurality of suitable marker or label species) in an immunochemical assay, such as an immunohistochemical or ELISA-type assay enhances the speed of reaction and the sensitivity (and, probably, the accuracy and reliability) of such assays. It is believed that the attachment, according to the present invention, of a plurality of antibody molecules (e.g. about 5, 10, 15, 20 or more) to the polymer carrier or backbone leads to, for example: (a) increased statistical probability of obtaining a plurality of antibody molecules having the correct steric conformation for satisfactory binding to the complementary immunological component (such as an antigen), and (b) increased strength of binding to the complementary immunological component.
It may also be mentioned here that similar advantages are also believed to be attainable in application of the invention in, e.g., hybridization techniques (vide supra), in that detection sensitivity and reliability of such procedures is to be expected to be significantly enhanced by employing an appropriate conjugate according to the invention comprising a plurality of marker species and, possibly, a plurality of probe molecules.
Fourthly, and as a more general aspect, in preparing conjugates according to the present invention which comprise two different types of attached molecular species, the divinyl-sulfone-based coupling chemistry employed in the context of the invention, notably in combination with the use, in a manner according to the invention, of a lyotropic salt in the attachment of these molecular species, makes possible a very wide variation in the numbers of and/or ratio between the two types of molecular species which are attached to the polymeric carrier molecule: As already outlined above, the ability to regulate the reactivity of free vinyl groups in reagents or conjugates of the invention by varying the pH makes it possible to establish a desired level of loading of the polymeric carrier (in relation to the available number of reactive vinyl groups) with one of the molecular species in question, after which the inherent reactivity of the remaining, unreacted vinyl groups can be suppressed by adjustment of the pH of the medium; if desired, the "intermediate" conjugate may then be subjected to one or more purification procedures, e.g. by chromatographic means, before proceeding to attach the second type of molecular species of interest. Not only is it thus possible to prepare well-characterized conjugates, but it is also possible to exert a high degree of control of the preparation process in a straight-forward manner.
Fifthly, it is believed that conjugates according to the invention based on certain preferred types of polymeric carrier molecules, viz. polymeric carrier molecules which are substantially linear, possess tissue structure penetration properties in spite of a relatively high total molecular weight.